This proposal seeks to identify key genes involved in regulating stem cell quiescence. Disruption of the balance between stem cells, proliferating progenitor cells and post-mitotic differentiated cells can result in a number of disease states including leukemias and myeloproliferative disorders. We hypothesize that cyclin-dependent kinase inhibitors (cdki's) such as p2l and p27 serve as gatekeepers in determining whether stem cells remain quiescent or begin to cycle. We have demonstrated that the expression of p21 is elevated in quiescent hematopoietic progenitor (CD34+lin- and CD34+ 5-FU-resistant) cells. We also have demonstrated subcellular redistribution of p27 upon activation of quiescent lymphocytes. We propose that artificial downmodulation of cdki's will induce quiescent stem cells to cycle. This proposal will 1. Establish the levels of the p21, p16, p27 and p57 cdki's in quiescent stem cells. This will enable these cell cycle inhibitors to be evaluated as candidate genes for maintaining stem cells in the quiescent state. The levels of these proteins individually and coordinately in stem cell candidates will be determined. Because no evaluation of cell cycle regulators in stem cells has yet been undertaken, the novel information gained through these experiments will establish genetic benchmarks for interpreting stem cell behavior. 2. Determine the changes in the subcellular distribution and expression of cdki's as cells exit from quiescence. We predict that the commitment by stem cells to self-renew or to give rise to progeny cells is likely to be associated with a decrease in the levels of cdki cell-cycle inhibitor proteins. In order to determine which protein changes induce stem cells to cycle, one first must establish how levels of these proteins and their distribution change as stem cells leave quiescence. 3. Determine whether cdki downmodulation is sufficient for recruitment of quiescent stem cells into the cycling pool. Establishing whether quiescence directly results from the activity of candidate cdki's requires functional confirmation. We will develop and test anti-sense approaches to modulate cdki expression levels in stem cells. The initial approach will be artificially to downmodulate p21 and measure whether this causes cells to leave quiescence. These experiments will apply recent findings in the molecular controls on cell growth with a central and long-standing question in cell biology--the balance between quiescence, self-renewal and maturation of stem cells. Insights gained from these studies should have a significant impact on gene therapy strategies and should generate novel tools for the study of leukemias and myelodysplastic syndromes.